A novel nucleic acid linker for multi-gene expression enhances plant and animal synthetic biology.

The production of compact vectors for gene stacking is hindered by a lack of effective linkers. Here, we report that a 26-nt nucleic acid linker, NAL1, from the fungus Glarea lozoyensis and its truncated derivatives could connect two genes as a bicistron, enabling independent translation in a maize protoplast transient expression system and human 293 T cells. The optimized 9-nt NAL10 linker was then used to connect four genes driven by a bidirectional promoter; this combination was successfully used to reconstruct the astaxanthin biosynthesis pathway in transgenic maize. The short and efficient nucleic acid linker NAL10 can be widely used in multi-gene expression and synthetic biology in animals and plants.

Identification and characterization of yellow stripe-like genes in maize suggest their roles in the uptake and transport of zinc and iron.

BACKGROUND: Yellow Stripe-Like (YSL) proteins are involved in the uptake and transport of metal ions. They play important roles in maintaining the zinc and iron homeostasis in Arabidopsis, rice (Oryza sativa), and barley (Hordeum vulgare). However, proteins in this family have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: In this study, we identified 19 ZmYSLs in the maize genome and analyzed their structural features. The results of a phylogenetic analysis showed that ZmYSLs are homologous to YSLs of Arabidopsis and rice, and these proteins are divided into four independent branches. Although their exons and introns have structural differences, the motif structure is relatively conserved. Analysis of the cis-regulatory elements in the promoters indicated that ZmYSLs might play a role in response to hypoxia and light. The results of RNA sequencing and quantitative real-time PCR analysis revealed that ZmYSLs are expressed in various tissues and respond differently to zinc and iron deficiency. The subcellular localization of ZmYSLs in the protoplast of maize mesophyll cells showed that they may function in the membrane system. CONCLUSIONS: This study provided important information for the further functional analysis of ZmYSL, especially in the spatio-temporal expression and adaptation to nutrient deficiency stress. Our findings provided important genes resources for the maize biofortification.

Maize Interveinal Chlorosis 1 links the Yang Cycle and Fe homeostasis through Nicotianamine biosynthesis.

The Yang cycle is involved in many essential metabolic pathways in plant growth and development. As extended products of the Yang cycle, the function and regulation network of ethylene and polyamines are well characterized. Nicotianamine (NA) is also a product of this cycle and works as a key metal chelator for iron (Fe) homeostasis in plants. However, interactions between the Yang cycle and NA biosynthesis remain unclear. Here, we cloned maize interveinal chlorosis 1 (mic1), encoding a 5'-methylthioadenosine nucleosidase (MTN), that is essential for 5'-methylthioadenosine (MTA) salvage and NA biosynthesis in maize (Zea mays). A single base G-A transition in the fourth exon of mic1 causes a Gly to Asp change, resulting in increased MTA, reduced Fe distribution, and growth retardation of seedlings. Knockout of ZmMIC1 but not its paralog ZmMTN2 by CRISPR/Cas9 causes interveinal chlorosis, indicating ZmMIC1 is mainly responsible for MTN activity in maize. Transcriptome analysis showed a typical response of Fe deficiency. However, metabolic analysis revealed dramatically reduced NA content in mic1, suggesting NA biosynthesis was impaired in the mutant. Exogenous application of NA transiently reversed the interveinal chlorosis phenotype of mic1 seedlings. Moreover, the mic1 mutant overexpressing a NA synthase gene not only recovered from interveinal chlorosis and growth retardation but was also fertile. These findings provide a link between the Yang cycle and NA biosynthesis, which highlights an aspect of Fe homeostasis regulation in maize.

Pentatricopeptide repeat protein CNS1 regulates maize mitochondrial complex III assembly and seed development.

Mitochondrial function relies on the assembly of electron transport chain complexes, which requires coordination between proteins encoded by the mitochondrion and those of the nucleus. Here, we cloned a maize (Zea mays) cytochrome c maturation FN stabilizer1 (CNS1) and found it encodes a pentatricopeptide repeat (PPR) protein. Members of the PPR family are widely distributed in plants and are associated with RNA metabolism in organelles. P-type PPR proteins play essential roles in stabilizing the 3'-end of RNA in mitochondria; whether a similar process exists for stabilizing the 5'-terminus of mitochondrial RNA remains unclear. The kernels of cns1 exhibited arrested embryo and endosperm development, whereas neither conventional splicing deficiency nor RNA editing difference in mitochondrial genes was observed. Instead, most of the ccmFN transcripts isolated from cns1 mutant plants were 5'-truncated and therefore lacked the start codon. Biochemical and molecular data demonstrated that CNS1 is a P-type PPR protein encoded by nuclear DNA and that it localizes to the mitochondrion. Also, one binding site of CNS1 located upstream of the start codon in the ccmFN transcript. Moreover, abnormal mitochondrial morphology and dramatic upregulation of alternative oxidase genes were observed in the mutant. Together, these results indicate that CNS1 is essential for reaching a suitable level of intact ccmFN transcripts through binding to the 5'-UTR of the RNAs and maintaining 5'-integrity, which is crucial for sustaining mitochondrial complex III function to ensure mitochondrial biogenesis and seed development in maize.

Novel prospects for scarless wound healing: The roles of myofibroblasts and adipocytes.

Disturbances or defects in the process of wound repair can disrupt the delicate balance of cells and molecules necessary for complete wound healing, thus leading to chronic wounds or fibrotic scars. Myofibroblasts are one of the most important cells involved in fibrotic scars, and reprogramming provides a potential avenue to increase myofibroblast clearance. Although myofibroblasts have long been recognized as terminally differentiated cells, recent studies have shown that myofibroblasts have the capacity to be reprogrammed into adipocytes. This review intends to summarize the potential of reprogramming myofibroblasts into adipocytes. We will discuss myofibroblast lineage tracing, as well as the known mechanisms underlying adipocyte regeneration from myofibroblasts. In addition, we investigated different changes in myofibroblast gene expression, transcriptional regulators, signalling pathways and epigenetic regulators during skin wound healing. In the future, myofibroblast reprogramming in wound healing will be better understood and appreciated, which may provide new ideas for the treatment of scarless wound healing.

Mediation of Zinc and Iron Accumulation in Maize by ZmIRT2, a Novel Iron-Regulated Transporter.

Iron (Fe) is an essential micronutrient for plant growth. Iron-regulated transporters (IRTs) play important roles in Fe2+ uptake and transport in strategy I plants. Maize (Zea mays) belongs to a strategy II plant, in which mugineic acid (MA)-Fe3+ uptake is mainly carried out by Yellow Stripe 1 (YS1). However, ZmIRT1 was previously identified by our laboratory. In this study, we isolated a novel gene from maize (ZmIRT2), which is highly homologous to OsIRT2 and ZmIRT1. ZmIRT2 was expressed in roots and anther and was induced by Fe and zinc (Zn) deficiencies. ZmIRT2-GFP fusion protein localized to the plasma membrane and endoplasmic reticulum. ZmIRT2 reversed growth defects involving Zn and Fe uptake in mutant yeast. ZmIRT2 overexpression in maize led to elevated Zn and Fe levels in roots, shoots and seeds of transgenic plants. Transcript levels of ZmIRT1 were elevated in roots, while levels of YS1 were reduced in shoots of ZmIRT2 transgenic plants. Our results imply that ZmIRT2 may function solely with ZmIRT1 to mediate Fe uptake in roots. ZmIRT1, ZmIRT2 and ZmYS1 may function in a cooperative manner to maintain Zn and Fe homeostasis in ZmIRT2 overexpressing plants. Furthermore, ZmIRT2 could be used in fortification efforts to elevate Zn and Fe levels in crop plants.

Genome-wide analysis of the NAAT, DMAS, TOM, and ENA gene families in maize suggests their roles in mediating iron homeostasis.

BACKGROUND: Nicotianamine (NA), 2'-deoxymugineic acid (DMA), and mugineic acid (MA) are chelators required for iron uptake and transport in plants. Nicotianamine aminotransferase (NAAT), 2'-deoxymugineic acid synthase (DMAS), transporter of MAs (TOM), and efflux transporter of NA (ENA) are involved in iron uptake and transport in rice (Oryza sativa), wheat (Triticum aestivum), and barley (Hordeum vulgare); however, these families have not been fully identified and comprehensively analyzed in maize (Zea mays L.). RESULTS: Here, we identified 5 ZmNAAT, 9 ZmDMAS, 11 ZmTOM, and 2 ZmENA genes by genome mining. RNA-sequencing and quantitative real-time PCR analysis revealed that these genes are expressed in various tissues and respond differently to high and low iron conditions. In particular, iron deficiency stimulated the expression of ZmDMAS1, ZmTOM1, ZmTOM3, and ZmENA1. Furthermore, we determined protein subcellular localization by transient expression of green fluorescent protein fusions in maize mesophyll protoplasts. ZmNAAT1, ZmNAAT-L4, ZmDMAS1, and ZmDMAS-L1 localized in the cytoplasm, whereas ZmTOMs and ZmENAs targeted to plasma and tonoplast membranes, endomembranes, and vesicles. CONCLUSIONS: Our results suggest that the different gene expression profiles and subcellular localizations of ZmNAAT, ZmDMAS, ZmTOM, and ZmENA family members may enable specific regulation of phytosiderophore metabolism in different tissues and under different external conditions, shedding light on iron homeostasis in maize and providing candidate genes for breeding iron-rich maize varieties.

Metabolic engineering of astaxanthin-rich maize and its use in the production of biofortified eggs.

Production of the high-value carotenoid astaxanthin, which is widely used in food and feed due to its strong antioxidant activity and colour, is less efficient in cereals than in model plants. Here, we report a new strategy for expressing ß-carotene ketolase and hydroxylase genes from algae, yeasts and flowering plants in the whole seed using a seed-specific bidirectional promoter. Engineered maize events were backcrossed to inbred maize lines with yellow endosperm to generate progenies that accumulate astaxanthin from 47.76 to 111.82 mg/kg DW in seeds, and the maximum level is approximately sixfold higher than those in previous reports (16.2-16.8 mg/kg DW) in cereals. A feeding trial with laying hens indicated that they could take up astaxanthin from the maize and accumulate it in egg yolks (12.10-14.15 mg/kg) without affecting egg production and quality, as observed using astaxanthin from Haematococcus pluvialis. Storage stability evaluation analysis showed that the optimal conditions for long-term storage of astaxanthin-rich maize are at 4 °C in the dark. This study shows that co-expressing of functional genes driven by seed-specific bidirectional promoter could dramatically boost astaxanthin biosynthesis in every parts of kernel including embryo, aleurone layer and starch endosperm other than previous reports in the starch endosperm only. And the staple crop maize could serve as a cost-effective plant factory for reliably producing astaxanthin.

ZmGRAS11, transactivated by Opaque2, positively regulates kernel size in maize.

Although the genetic basis for endosperm development in maize (Zea mays) has been well studied, the mechanism for coordinating grain filling with increasing kernel size remains elusive. Here, we report that increased kernel size was selected during modern breeding and identify a novel DELLA-like transcriptional regulator, ZmGRAS11, which positively regulates kernel size and kernel weight in maize. We find that Opaque2, a core transcription factor for zein protein and starch accumulation, transactivates the expression of ZmGRAS11. Our data suggest that the Opaque2-ZmGRAS11 module mediates synergistic endosperm enlargement with grain filling.

Optimization of extraction of total trans-resveratrol from peanut seeds and its determination by HPLC.

Resveratrol, a stilbene phytoalexin in plants, is believed to benefit human health. In this study, an optimized enzyme-assisted method was developed to extract the total content of trans-resveratrol (free or combined with glucose) in peanut seeds, followed by detection using high-performance liquid chromatography. The extraction process was optimized by Box-Behnken design and response surface methodology. The optimized enzyme concentration, digestion time, pH, and temperature were 3.02 g/L, 57.06 min, 5.88, and 51.05°C, respectively. Validation tests indicated that the experimental yield of trans-resveratrol was 0.183 ± 0.007 µg/g with a relative standard deviation of 3.87% (n = 5) under the optimal condition, which was closely agreed with the predicted value (0.182 µg/g). The recoveries obtained from the spiked samples were varied from 89.4 to 103.9%. Therefore, this study will provide a useful method for quantification of total trans-resveratrol in peanut seeds.

Improving Zinc and Iron Accumulation in Maize Grains Using the Zinc and Iron Transporter ZmZIP5.

Zinc (Zn) and iron (Fe) are essential micronutrients for plant growth. Thus, it is important to understand the mechanisms of uptake, transport and accumulation of these micronutrients in maize to improve crop nutritional quality. Members of the zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) family are responsible for the uptake and transport of divalent metal ions in plant. Previously, we showed that ZmZIP5 functionally complemented the Zn uptake double mutant zrt1zrt2, Fe-uptake double mutant fet3fet4 in yeast. In our ß-glucuronidase (GUS) assay, the germinated seeds, young sheaths, and stems of ZmZIP5-promoter-GUS transgenic plants were stained. We generated and compared two maize lines for this study: Ubi-ZmZIP5, in which ZmZIP5 was constitutively overexpressed, and ZmZIP5i, a RNAi line. At the seedling stage, high levels of Zn and Fe were found in the roots and shoots of Ubi-ZmZIP5 plants, whereas low levels were found in the ZmZIP5i plants. Zn and Fe contents decreased in the seeds of Ubi-ZmZIP5 plants and remained unchanged in the seeds of ZmZIP5i plants. The seeds of Leg-ZmZIP5 plants, in which ZmZIP5 overexpression is specific to the endosperm, had higher levels of Zn and Fe. Our results imply that ZmZIP5 may play a role in Zn and Fe uptake and root-to-shoot translocation. Endosperm-specific ZmZIP5 overexpression could be useful for Zn and Fe biofortification of cereal grains.

Genome-scale mining of root-preferential genes from maize and characterization of their promoter activity.

BACKGROUND: Modification of root architecture and improvement of root resistance to stresses can increase crop productivity. Functional analyses of root-specific genes are necessary for root system improvement, and root-specific promoters enable research into the regulation of root development and genetic manipulation of root traits. Maize is an important crop species; however, little systematic mining of root-specific genes and promoters has been performed to date. RESULTS: Genomic-scale mining based on microarray data sets followed by transcript detection resulted in the identification of 222 root-specific genes. Gene Ontology enrichment analyses revealed that these 222 root-specific genes were mainly involved in responses to chemical, biotic, and abiotic stresses. Of the 222 genes, 33 were verified by quantitative reverse transcription polymerase chain reaction, and 31 showed root-preferential activity. About 2 kb upstream 5 of the 31 identified root-preferential genes were cloned from the maize genome as putative promoters and named p8463, p5023, p1534, p8531 and p6629. GUS staining of transgenic maize-derived promoter-GUS constructs revealed that the five promoters drove GUS expression in a root-preferential manner. CONCLUSIONS: We mined root-preferential genes and their promoters in maize and verified p8463, p5023, p1534, p8531 and p6629 as root-preferential promoters. Our research enables the identification of other tissue-specific genes and promoters in maize and other species. In addition, the five promoters may enable enhancement of target gene(s) of maize in a root-preferential manner to generate novel maize cultivars with resistance to water, fertilizer constraints, or biotic stresses.

Synthesis of Seed-Specific Bidirectional Promoters for Metabolic Engineering of Anthocyanin-Rich Maize.

Tissue-specific promoters play an important role in plant molecular farming. Here, we describe a strategy to modify the tissue specificity of a maize embryo-specific bidirectional promoter PZmBD1. Six types of cis-elements, i.e. RY repeats (R), GCN4 (G), the prolamin box (P), Skn-1 (S), and the ACGT and AACA (A) motifs, were collected and fused to PZmBD1 to generate eight chimeric putative bidirectional promoters. Qualitative and quantitative analysis of reporter genes driven by the promoters showed that two promoters exhibited high seed-specific bidirectional activity in maize transient and stable transformed systems. The stronger one was chosen and fused to the intergenic region of two gene clusters consisting of four anthocyanin biosynthesis-related genes (ZmBz1, ZmBz2, ZmC1 and ZmR2) and seven reporter genes, resulting in the first embryo and endosperm anthocyanin-rich purple maize. Anthocyanin analysis showed that the total anthocyanin content reaches 2,910 mg kg-1 DW in transgenic maize and cyanidin is the major anthocyanin in transgenic maize, as in natural varieties. The expression profile analysis of endogenous genes showed that the anthocyanin biosynthesis pathway was activated by two transgenic transcription factor genes ZmC1 and ZmR2. Our results indicate that both the modification strategy and these functionally characterized tissue-specific bidirectional promoters generated could be used for genetic research and development of plant biotechnology products. The anthocyanin-rich purple maize could provide economic natural colorants for the food and beverage industry, and valuable germplasm for developing anthocyanin-rich fresh corn.

Calcium-dependent protein kinase 21 phosphorylates 14-3-3 proteins in response to ABA signaling and salt stress in rice.

The calcium-dependent protein kinases (CDPKs) are a class of plant-specific kinase that directly bind Ca2+ and mediate the calcium-signaling pathways to play important physiological roles in growth and development. The rice genome contains 31 CDPK genes, one of which, OsCPK21, is known to modulate the abscisic acid (ABA) and salt stress responses in this crop; however, the molecular mechanisms underlying this regulation are largely unknown. In the present study, we performed yeast two-hybrid screening, glutathione S-transferase pull-down, co-immunoprecipitation, and bimolecular fluorescence complementation assays to confirm the interaction between OsCPK21 and one of its putative targets, Os14-3-3 (OsGF14e). We used an in vitro kinase assay and site-directed mutagenesis to verify that OsCPK21 phosphorylates OsGF14e at Tyr-138. We used real-time PCR to reveal that several ABA and salt inducible genes were more highly expressed in the OsCPK21-OE and OsGF14e WT-OE plants than in the mutant OsGF14e Y138A-OE and wild-type plants. These results suggest that OsCPK21 phosphorylates OsGF14e to facilitate the response to ABA and salt stress.

Analysis of weighted co-regulatory networks in maize provides insights into new genes and regulatory mechanisms related to inositol phosphate metabolism.

BACKGROUND: D-myo-inositol phosphates (IPs) are a series of phosphate esters. Myo-inositol hexakisphosphate (phytic acid, IP6) is the most abundant IP and has negative effects on animal and human nutrition. IPs play important roles in plant development, stress responses, and signal transduction. However, the metabolic pathways and possible regulatory mechanisms of IPs in maize are unclear. In this study, the B73 (high in phytic acid) and Qi319 (low in phytic acid) lines were selected for RNA-Seq analysis from 427 inbred lines based on a screening of IP levels. By integrating the metabolite data with the RNA-Seq data at three different kernel developmental stages (12, 21 and 30 days after pollination), co-regulatory networks were constructed to explore IP metabolism and its interactions with other pathways. RESULTS: Differentially expressed gene analyses showed that the expression of MIPS and ITPK was related to differences in IP metabolism in Qi319 and B73. Moreover, WRKY and ethylene-responsive transcription factors (TFs) were common among the differentially expressed TFs, and are likely to be involved in the regulation of IP metabolism. Six co-regulatory networks were constructed, and three were chosen for further analysis. Based on network analyses, we proposed that the GA pathway interacts with the IP pathway through the ubiquitination pathway, and that Ca(2+) signaling functions as a bridge between IPs and other pathways. IP pools were found to be transported by specific ATP-binding cassette (ABC) transporters. Finally, three candidate genes (Mf3, DH2 and CB5) were identified and validated using Arabidopsis lines with mutations in orthologous genes or RNA interference (RNAi)-transgenic maize lines. Some mutant or RNAi lines exhibited seeds with a low-phytic-acid phenotype, indicating perturbation of IP metabolism. Mf3 likely encodes an enzyme involved in IP synthesis, DH2 encodes a transporter responsible for IP transport across organs and CB5 encodes a transporter involved in IP co-transport into vesicles. CONCLUSIONS: This study provides new insights into IP metabolism and regulation, and facilitates our development of a better understanding of the functions of IPs and how they interact with other pathways involved in plant development and stress responses. Three new genes were discovered and preliminarily validated, thereby increasing our knowledge of IP metabolism.

The intergenic region of the maize defensin-like protein genes Def1 and Def2 functions as an embryo-specific asymmetric bidirectional promoter.

Bidirectional promoters are identified in diverse organisms with widely varied genome sizes, including bacteria, yeast, mammals, and plants. However, little research has been done on any individual endogenous bidirectional promoter from plants. Here, we describe a promoter positioned in the intergenic region of two defensin-like protein genes, Def1 and Def2 in maize (Zea mays). We examined the expression profiles of Def1 and Def2 in 14 maize tissues by qRT-PCR, and the results showed that this gene pair was expressed abundantly and specifically in seeds. When fused to either green fluorescent protein (GFP) or ß-glucuronidase (GUS) reporter genes, P ZmBD1 , P ZmDef1 , and P ZmDef2 were active and reproduced the expression patterns of both Def1 and Def2 genes in transformed immature maize embryos, as well as in developing seeds of transgenic maize. Comparative analysis revealed that PZmBD1 shared most of the expression characteristics of the two polar promoters, but displayed more stringent embryo specificity, delayed expression initiation, and asymmetric promoter activity. Moreover, a truncated promoter study revealed that the core promoters only exhibit basic bidirectional activity, while interacting with necessary cis-elements, which leads to polarity and different strengths. The sophisticated interaction or counteraction between the core promoter and cis-elements may potentially regulate bidirectional promoters.

Isolation of a maize ZmCI-1B promoter and characterization of its activity in transgenic maize and tobacco.

KEY MESSAGE: The 2-kb ZmCI - 1B promoter is active in the root and embryo and induced by wounding in maize and the 220-bp 5'-deleted segment maybe the minimal promoter. The subtilisin-chymotrypsin inhibitor gene, CI-1B of Zea mays (ZmCI-1B), has been suggested to induce the maize defense system to resist insect attack. Real-time RT-PCR showed that ZmCI-1B gene exhibited especially high expression in roots and embryos. The 2-kb full-length promoter of ZmCI-1B gene was isolated from the maize genome and used to drive expression of a beta-glucuronidase (GUS) reporter gene for transient expression and stable expression analysis in maize. The results of GUS histochemical staining in transgenic maize plants revealed that the ZmCI-1B promoter induced GUS expression preferentially in roots and embryos and in response to wounding. A series of 5'-deleted segments of the ZmCI-1B promoter were cloned individually to drive GUS expression for further analysis. Deletion analysis combined with the histochemical staining of transgenic tobacco plants revealed 220-bp segment could drive GUS in a tissue-specific and wounding-induced expression in tobacco; thus, it maybe the minimally active promoter of ZmCI-1B gene. Furthermore, it revealed that the ZmCI-1B promoter contained tissue-specific and wounding-induced elements.

Identification and functional characterization of bidirectional gene pairs and their intergenic regions in maize.

BACKGROUND: Bidirectional gene pairs exist as a specific form of gene organization in microorganisms and mammals as well as in model plant species, such as Arabidopsis and rice. Little is known about bidirectional gene pairs in maize, which has a large genome and is one of the most important grain crops. RESULTS: We conducted a genome-wide search in maize using genome sequencing results from the inbred line B73. In total, 1696 bidirectional transcript pairs were identified using a modified search model. We functionally characterized the promoter activity of the intergenic regions of most of the bidirectional transcript pairs that were expressed in embryos using a maize embryo transient expression system. A comparative study of bidirectional gene pairs performed for three monocot (Zea mays, Sorghum bicolor and Oryza sativa) and two dicot (Arabidopsis thaliana and Glycine max) plant genomes showed that bidirectional gene pairs were abundant in the five plant species. Orthologous bidirectional gene pairs were clearly distinguishable between the monocot and dicot species although the total numbers of orthologous bidirectional genes were similar. Analysis of the gene pairs using the Blast2GO software suite showed that the molecular functions (MF), cellular components (CC), and biological processes (BP) associated with the bidirectional transcripts were similar among the five plant species. CONCLUSIONS: The evolutionary analysis of the function and structure of orthologous bidirectional gene pairs in various plant species revealed a potential pathway of their origin, which may be required for the evolution of a new species.

Identification and characterization of promoters specifically and strongly expressed in maize embryos.

The use of maize seeds as bioreactors has several advantages for the production of recombinant proteins in plant biotechnology, but available embryo-specific and strong promoters are limited. Here, we describe a genome-scale microarray-based approach to identify embryo-specifically and strongly expressed genes and their promoters in maize. We identified 28 embryo-preferred and abundantly expressed genes based on our microarray data. These embryo-preferred genes were further analysed using the UniGene database and by quantitative reverse transcriptase-PCR leading to the identification of seven genes (Zm.2098, Zm.13387, Zm.66589, Zm.85502, Zm.68129, Zm.3896 and Zm.2941) as embryo-specific genes with higher expression levels relative to maize globulin-1. The putative promoters of five embryo-specific genes (Zm.13387, Zm.66589, Zm.85502, Zm.3896 and Zm.2941) were isolated and all exhibited strong promoter activities when transiently expressed in maize embryos of 20 DAP. The embryo specificity and expression levels of the promoters of four genes (Zm.13387, Zm.85502, Zm.3896 and Zm.2941) were further examined in transgenic maize plants, revealing that they are strong promoters in embryos of all four developmental stages tested compared with reference globulin-1 promoter. Moreover, Zm.2941 and Zm.3896 promoters are stringently embryo-specific promoters, while Zm.85502 promoter is basically embryo specific yet wounding inducible in non-seed tissues, and Zm.13387 promoter is developmentally expressed in both embryo and aleurone with wounding-induced activity in non-seed tissues. Our study provides novel embryo-specific and strong promoters that are suitable for production of high-level recombinant proteins in maize embryos.